Method for the treatment or prevention of Flaviviridea viral infection using nucleoside analogues

ABSTRACT

In accordance with the present invention there is provided a method for treating or preventing a  Flaviviridea  viral infection in a host comprising administering a therapeutically effective amount of at least one compound of formula (I) or (II)  
                 
 
     or  
     or a pharmaceutically acceptable salts thereof, wherein Ra, R, Z and Y are defined in the application.

[0001] This application claims priority from U.S. application Ser. No.60/163,405 and U.S. application Ser. No. 60/163,394 filed Nov. 4, 1999which are herein incorporated by reference

FIELD OF THE INVENTION

[0002] The present invention relates to a method for the treatment orprevention Flaviviridae viral infection using nucleoside analogues.

BACKGROUND OF THE INVENTION

[0003] Hepatitis is a disease occurring throughout the world. It isgenerally of viral nature, although there are other causes known. Viralhepatitis is by far the most common form of hepatitis. Nearly 750,000Americans are affected by hepatitis each year, and out of those, morethan 150,000 are infected with the hepatitis C virus (HCV).

[0004] HCV is a positive-stranded RNA virus belonging to theFlaviviridae family and has closest relationship to the pestivirusesthat include hog cholera virus and bovine viral diarrhea virus (BVDV).HCV is believed to replicate through the production of a complementarynegative-strand RNA template. Due to the lack of efficient culturereplication system for the virus, HCV particles were isolated frompooled human plasma and shown, by electron microscopy, to have adiameter of about 50-60 nm. The HCV genome is a single-stranded,positive-sense RNA of about 9,600 bp coding for a polyprotein of3009-3030 amino-acids, which is cleaved, post-translationally bycellular and two viral proteinases into mature viral proteins (core, E1,E2, p7, NS2, NS3, NS4A, NS4B, NS5A/ NS5B). It is believed that thestructural proteins, E1 and E2, the major glycoproteins are embeddedinto a viral lipid envelop and form stable heterodimers. The structuralcore protein interacts with the viral RNA genome to form thenucleocapsid. The genes or regions designated NS2 to NS5 code fornonstructural proteins some of which have enzymatic functions involvedin virus replication and protein processing including a polymerase,protease and helicase.

[0005] The main sources of contamination with HCV is blood. Themagnitude of the HCV infection as a health problem is illustrated by theprevalence among high-risk groups. For example, 60% to 90% ofhemophiliacs and more than 80% of intravenous drug abusers in westerncountries are chronically infected with HCV. For intravenous drugabusers, the prevalence varies from about 28% to 70% depending on thepopulation studied. The proportion of new HCV infections associated withpost-transfusion has been markedly reduced lately due to advances indiagnostic tools used to screen blood donors.

[0006] The treatment currently available for HCV infection is interferon(IFN), ribavirin, and a combinaison of the two (REBETRON). However,according to different clinical studies, only 70% of treated patientsnormalize alanine aminotransferase (ALT) levels in the serum and afterdiscontinuation of IFN, 35% to 45% of these responders relapse. Ingeneral, only 20% to 25% of patients have long-term responses to IFN. Onthe other hand, pilot studies suggest that combination treatment withIFN plus Ribavirin (RIBA) results in sustained response in the majorityof patients. Different genotypes of HCV respond differently to IFNtherapy, genotype 1b is more resistant to IFN therapy than type 2 and 3.

[0007] There is therefore a great need for the development oftherapeutic agent for treating or preventing Flaviviridae viralinfection.

SUMMARY OF THE INVENTION

[0008] The present invention provides a method for treating orpreventing a Flaviviridea viral infection in a host comprisingadministering a therapeutically effective amount of at least onecompound selected from:

or

[0009] or a pharmaceutically acceptable salts thereof,

[0010] wherein:

[0011] R is H, —NR₂R₃ or OR₄ wherein

[0012] R₂ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₈cycloalkyl;

[0013] R₃ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl;

[0014] R₄ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl; and

[0015] Z is H, OR_(4′), or —NR_(2′)R_(3′) wherein:

[0016] R_(2′) is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₈cycloalkyl,

[0017] R_(3′) is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl;

[0018] R_(4′) is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl; and

[0019] and;

[0020] Y is N or C—X;

[0021] X is chosen from of H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, CN, CF₃, N₃, NO₂, C₆₋₁₀ aryl,. C₆₋₁₀ heteroaryl and CORb

[0022] wherein Rb is chosen from of H, OH, SH, C₁₋₆ alkyl, C₁₋₆

[0023] aminoalkyl, C₁₋₆ alkoxy and C₁₋₆ thioalkyl;

[0024] and Ra is chosen from of H, monophosphate, diphosphate,triphosphate, carbonyl substituted with a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₆₋₁₀ aryl, and

[0025] wherein each Rc are independently chosen from H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and an hydroxy protecting group,

[0026] wherein said nucleoside is present in the form of the (−)enantiomer, the (+) enantiomer or mixtures thereof, including racemicmixtures.

[0027] The present invention provides a method for treating aFlaviviridea viral infection in a host comprising administering atherapeutically effective amount of at least one compound as describedin the present application.

[0028] The present invention provides a method for preventing aFlaviviridea viral infection in a host comprising administering atherapeutically effective amount of at least one compound as describedin the present application.

[0029] The present invention provides a method for reducing thebiological activity of a Flaviviridea viral infection in a hostcomprising administering a therapeutically effective amount of at leastone compound as described in the present application.

[0030] In another aspect, there is provided a pharmaceutical formulationcomprising the compound of the invention in combination with apharmaceutically acceptable carrier or excipient.

[0031] Still another aspect, there is provided a method for treating orpreventing a Flaviviridea viral infection in a host comprisingadministering to the subject a combination comprising at least onecompound according to formula I or formula II and at least one furthertherapeutic agent.

[0032] In another aspect of the invention is the use of a compoundaccording to formula I, for the preparation of a medicament for treatingor preventing a viral infections in a host.

[0033] In another aspect of the invention is the use of a compoundaccording to formula II, for the preparation of a medicament fortreating or preventing a viral infection in a host.

DETAILED DESCRIPTION OF THE INVENTION

[0034] In one embodiment, the viral infection is chosen fromFlaviviridea viral infections.

[0035] In one embodiment, the Flaviviridea viral infection is chosenfrom of Hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), hogcholera and yellow fever.

[0036] In an other embodiment, the Flaviviridea viral infection isHepatitis C.

[0037] In one embodiment, there is also provided a method for inhibitingor reducing the activity of viral polymerase in a host, when compared toa standard comprising administering a therapeutically effective amountof a compound having the formula I.

[0038] In one embodiment, there is also provided a method for inhibitingor reducing the activity of viral polymerase in a host, when compared toa standard, comprising administering a therapeutically effective amountof a compound having the formula II.

[0039] In one-embodiment, there is also provided a method for inhibitingthe activity of viral polymerase in a host, when compared to a standardcomprising administering a therapeutically effective amount of acompound having the formula I.

[0040] In one embodiment, there is also provided a method for inhibitingthe activity of viral polymerase in a host, when compared to a standard,comprising administering a therapeutically effective amount of acompound having the formula II.

[0041] In one embodiment, there is also provided a method for reducingthe activity of viral polymerase in a host, when compared to a standardcomprising administering a therapeutically effective amount of acompound having the formula I.

[0042] In one embodiment, there is also provided a method for reducingthe activity of viral polymerase in a host, when compared to a standard,comprising administering a therapeutically effective amount of acompound having the formula II.

[0043] In another embodiment, the viral polymerase is HCV polymerase.

[0044] In one embodiment, the compounds and methods of the presentinvention comprise those wherein the following embodiments are present,either independently or in combination.

[0045] In one embodiment, R₄ is C₁₋₆alkyl.

[0046] In one embodiment, R₄ is methyl or ethyl.

[0047] In one embodiment, R is NH₂, H or OH.

[0048] In a further embodiment R is NH₂ or OH.

[0049] In a further embodiment R is OH.

[0050] In one embodiment R is —NR₂R₃ wherein R₂ is C₃₋₈cycloalkyl and R₃is H.

[0051] In one embodiment R is —NR₂R₃ wherein R₂ is C₃₋₅cycloalkyl and R₃is H.

[0052] In one embodiment R is —NR₂R₃ wherein R₂ is cyclopropyl orcyclopentyl and R₃ is H.

[0053] In one embodiment R is —NR₂R₃ wherein R₂ is cycloropyl and R₃ isH.

[0054] In one embodiment Ra is chosen from H, monophosphate,diphosphate, and triphosphate.

[0055] In one embodiment Ra is chosen from monophosphate, diphosphate,and triphosphate.

[0056] In one embodiment Ra is triphosphate.

[0057] In one embodiment Ra is H.

[0058] In one embodiment, Z is H, OH, —NR_(2′)R_(3′) wherein:

[0059] R_(2′) is H, or C₁₋₆ alkyl;and

[0060] R_(3′) is H, or C₁₋₆ alkyl.

[0061] In one embodiment, R_(4′) is C₁₋₆alkyl.

[0062] In one embodiment, R_(4′) is methyl or ethyl.

[0063] In one embodiment Z is chosen from OH or NH₂.

[0064] In one embodiment Z is OH.

[0065] In another embodiment Z is NH₂.

[0066] In another embodiment, Y is N.

[0067] In another embodiment, Y is C—X.

[0068] In another embodiment, X is H, C₁₋₆alkyl or halogen.

[0069] In another embodiment, X is H, methyl or halogen.

[0070] In another embodiment, X is methyl, —HC═CH₂ and —C≡H

[0071] In another embodiment, X is H or halogen.

[0072] In another embodiment, X is halogen.

[0073] In another embodiment, X is H, methyl or F.

[0074] In another embodiment, X is H or F.

[0075] In another embodiment, X is H.

[0076] In another embodiment, X is F.

[0077] It will be appreciated by those skilled in the art that thecompounds of formula (I) contain at least two chiral centers which aremarked by an asterisk (*) on the general formula (I) or (II) Thecompounds of formula (I) and (II) thus exist in the form of twodifferent optical isomers (i.e. (+) or (−) enantiomers or β-L and β-D).All such enantiomers and mixtures thereof including racemic mixtures areincluded within the scope of the invention.

[0078] The single optical isomer or enantiomer can be obtained by methodwell known in the art, such as chiral synthesis, chiral HPLC, enzymaticresolution and chiral auxiliary.

[0079] Compounds of the invention include, for compounds having thestructure of the formula I:

[0080] Compound 1cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;Compound 1 (−)(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;; Compound 1 (+)(+)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane

[0081] Compound 2cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane;Compound 2 (−)(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolaneCompound 2 (+)(+)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane

[0082] Compound 3cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 3 (−)(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 3 (+)(+)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane

[0083] Compound 4-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 4 (−)(−)-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 4 (+)(+)-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane

[0084] Compound 5 cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane;Compound 5 (−) (−)-cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane;Compound.5 (+) (+)-cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane

[0085] Compound 6 cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane;Compound 6 (−) (−)-cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane;Compound 6 (+) (+)-cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane

[0086] Compound 7cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane;Compound 7 (−)(−)-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane;Compound 7 (+)(+)-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane

[0087] Compound 8cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane; Compound 8(−) (−)-cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane;Compound 8 (+)(+)-cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane

[0088] In a further aspect of the present invention compounds of theinvention include

[0089] Compound 1 (−)(−)-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane

[0090] Compound 2 (−)(−)-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane

[0091] Compound 3 (−)(−)-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane

[0092] Compound 4 (−)(−)-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane

[0093] Compound 5 (−)(−)-cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane

[0094] Compound 6 (−)(−)-cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane

[0095] Compound 7 (−)(−)-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane

[0096] Compound 8 (−) (−)cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-cyclopropylamino-purine-9′-yl)-1,3-dioxolane

[0097] In a further embodiment, the compounds of the present inventionhave a triphosphate group in the 5′ position. In a further aspect of thepresent invention, the compounds of formula (I) are represented by:

[0098] Compound 9triphoshatecis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;Compound 9(−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;; Compound 9(+) (+)-triphosphate-cis-2-hydroxyethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane

[0099] Compound 10 Triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane;Compound 10 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolaneCompound 10 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane

[0100] Compound 11 Triphosphatecis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 11 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 11 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane

[0101] Compound 12 triphosphatecis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 12 (−) (−) -triphosphate-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 12 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane

[0102] Compound 135 Triphosphatecis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane; Compound 135 (−)(−)- triphosphate -cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane;Compound 13 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane

[0103] Compound 14 Triphosphate-cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane; Compound 14 (−)(−)-triphosphate -cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane;Compound 14 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane

[0104] Compound 15 Triphosphate-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane;Compound 15 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane;Compound 15 (+) (+)-triphosphate-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane

[0105] Compound 16 Triphosphate-cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane; Compound 16(−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane; Compound 16(+) (+)-triphosphate-cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane

[0106] In a further aspect of the present invention compounds of theinvention include:

[0107] Compound 9 (−)(−)-triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane

[0108] Compound 10 (−) triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane

[0109] Compound 11 (−) (−)- triphosphate-cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane

[0110] Compound 12 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane

[0111] Compound 13 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(guanin-9′-yl) -1,3-dioxolane

[0112] Compound 14 (−) (−)-triphasphate-cis-2-hydroxymethyl-4-(adenin-9′-yl) -1,3-dioxolane

[0113] Compound 15 (−) (−)-triphosphate-cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane

[0114] Compound 16 (−) (−)-triphosphatecis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane

[0115] In a further embodiment of the present invention the compounds offormula (II) include:

[0116] In one embodiment, a compound of formula II iscis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane (compound#17)

[0117] In one embodiment, a compound of formula II is(-)-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane (β-L-OddC)(compound#17 (−)).

[0118] In one embodiment, a compound of formula II is(+)-cis-2-hydroxymethyl-4-(cytosin-1′-yl,)-1,3-dioxolane (β-D-OddC)(compound#17 (+)).

[0119] In another embodiment, a compound of formula II iscis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane (5FOddC)(compound#18).

[0120] In another embodiment, a compound of formula II is(−)-cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane(β-L-5FOddC) (compound#18 (−)).

[0121] In another embodiment, a compound of formula II is(+)-cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane(β-D-5FOddC) (compound#18 (+)).

[0122] In another embodiment, a compound of formula II iscis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane (compound#19)

[0123] In another embodiment, a compound of formula II is(−)-cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane(compound#19 (−)).

[0124] In another embodiment, a compound of formula II is(+)-cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane(compound#19 (+)).

[0125] In another embodiment, a compound of formula II iscis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#20).

[0126] In another embodiment, a compound of formula II is(−)-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#20 (−)).

[0127] In another embodiment, a compound of formula II is(+)-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#20 (+)).

[0128] In another embodiment, a compound of formula II iscis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane (compound#21).

[0129] In another embodiment, a compound of formula II is(−)-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane (compound#21(−)).

[0130] In another embodiment, a compound of formula II is(+)-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane (compound#21(+)).

[0131] In a further embodiment, the compounds of the present inventionhave a triphosphate group in the 5′ position. In a further aspect of thepresent invention, the compounds of formula (II) are represented by:

[0132] In one embodiment, a compound of formula II istriphosphate-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane(β-OddC-tp) (compound#22).

[0133] In one embodiment, a compound of formula II is(−)-triphosphate-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane(β-L-OddC-tp) (compound#22 (−)).

[0134] In one embodiment, a compound of formula II is(+)-triphosphate-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane(β-D-OddC-tp) (compound#22 (+)).

[0135] In another embodiment, a compound of formula II istriphosphate-cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane(β-5FOddC-tp) (compound#23).

[0136] In another embodiment, a compound of formula II is(−)-triphosphate-cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane(β-L-5FOddC-tp) (compound#23 (−)).

[0137] In another embodiment, a compound of formula II is(+)-triphosphate-cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-dioxolane(β-D-5FOddC-tp) (compound#23 (+)).

[0138] In another embodiment, a compound of formula II istriphosphate-cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane(compound#24).

[0139] In another embodiment, a compound of formula II is(−)-triphosphate-cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane(compound#24 (−)).

[0140] In another embodiment, a compound of formula II is(+)-triphosphate-cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-dioxolane(compound#24 (+).

[0141] In another embodiment, a compound of formula II istriphosphate-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#25).

[0142] In another embodiment, a compound of formula II is(−)-triphosphate-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)a-1,3-dioxolane(compound#25 (−)).

[0143] In another embodiment, a compound of formula II is(+)-triphosphate-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#25 (+)).

[0144] In another embodiment, a compound of formula II istriphosphate-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane(compound#26).

[0145] In another embodiment, a compound of formula II is(−)-triphosphate-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane(compound#26 (−)).

[0146] In another embodiment, a compound of formula II is(+)-triphosphate-cis-2-hydroxymethyl-4-(N-1′-thiminyl)-1,3-dioxolane(compound#26 (+)).

[0147] In one embodiment the compounds of the present invention areprovided in the form of a single enantiomer at least 95% free of thecorresponding enantiomer.

[0148] In one embodiment the compounds of the present invention areprovided in the form of a single enantiomer at least 95% free of thecorresponding enantiomer.

[0149] In one embodiment the compounds of the present invention areprovided in the form of a single enantiomer at least 99% free of thecorresponding enantiomer.

[0150] In an other embodiment the compound of the present invention arein the form of the (+) enantiomer at least 95% free of the corresponding(−) enantiomer.

[0151] In an other embodiment the compound of the present invention arein the form of the (+) enantiomer at least 97% free of the corresponding(−) enantiomer.

[0152] In an other embodiment the compound of the present invention arein the form of the (+) enantiomer at least 99% free of the corresponding(−) enantiomer.

[0153] In an other embodiment embodiment, the compound of the presentinvention are in the form of the (−) enantiomer at least 95% free of thecorresponding (+) enantiomer.

[0154] In an other embodiment the compound of the present invention arein the form of the (−) enantiomer at least 97% free of the corresponding(+) enantiomer.

[0155] In an other embodiment the compound of the present invention arein the form of the (−) enantiomer at least 99% free of the corresponding(+) enantiomer.

[0156] There is also provided a pharmaceutically acceptable salts of thepresent invention. By the term pharmaceutically acceptable salts ofcompounds of general formula (I) and (II) are meant those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acids include hydrochloric, hydrobromic, sulphuric,nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic,salicylic, succinic, toleune-p-sulphonic, tartaric, acetic, citric,methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic andbenzenesulphonic acids. Other acids such as oxalic, while not inthemselves pharmaceutically acceptable, may be useful as intermediatesin obtaining the compounds of the invention and their pharmaceuticallyacceptable acid addition salts.

[0157] Salts derived from appropriate bases include alkali metal (e.g.sodium), alkaline earth metal (e.g. magnesium) ammonium and NR₄₊ (whereR is C₁₋₄ alkyl) salts.

[0158] References hereinafter to a compound according to the inventionincludes compounds of the general formula (I) and (II) and therepharmaceutically acceptable salts.

[0159] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

[0160] As used in this application, the term “alkyl” represents anunsubstituted or substituted (by a halogen, nitro, CONH₂, COOH, O—C₁₋₆alkyl, O—C₂₋₆ alkenyl, O—C₂₋₆ alkynyl, hydroxyl, amino, or COOQ, whereinQ is C₁₋₆ alkyl; C₂₋₆ alkenyl; C₂₋₆ alkynyl) straight chain, branchedchain or cyclic hydrocarbon moiety (e.g. isopropyl, ethyl, fluorohexylor cyclopropyl). The term alkyl is also meant to include alkyls in whichone or more hydrogen atoms is replaced by an halogen, more preferably,the halogen is fluoro (e.g. CF₃- or CF₃CH₂-).

[0161] The terms “alkenyl” and “alkynyl” represent an alkyl containingat least one unsaturated group (e.g. allyl).

[0162] The term “cycloalkyl” represents an alkyl which is cyclic, suchas cyclopropyl, cyclopentyl or cyclobutyl.

[0163] The term “hydroxy protecting group” is well known in the field oforganic chemistry. Such protecting groups may be found in T. Greene,Protective Groups In Organic Synthesis, (John Wiley & Sons, 1981).Example of hydroxy protecting groups include but are not limited toacetyl-2-thioethyl ester, pivaloyloxymethyl ester andisopropyloxycarbonyloxymethyl ester.

[0164] The term “aryl” represent an unsaturated carbocyclic moiety,optionally mono- or di-substituted with OH, SH, amino, halogen or C₁₋₆alkyl.

[0165] The term “heteroaryl” represent an aryl wherein at least onecarbon ring atom is substituted by an heteroatom (e.g. N, O, or S).

[0166] The term “aminoalkyl” represent an alkyl which is covalentlybonded to the adjacent atom through a nitrogen atom.

[0167] The term “thioalkyl” represent an alkyl which is covalentlybonded to the adjacent atom through a sulfur atom.

[0168] The term “alkoxy” represent an alkyl which is covalently bondedto the adjacent atom through an oxygen atom.

[0169] When there is a sulfur atom present, the sulfur atom can be atdifferent oxydation level, S, SO, or SO₂. All such oxydation level arewithin the scope of the present invention.

[0170] The term “host” represent any mammals including humans.

[0171] In one embodiment, the host is human.

[0172] The compounds of the present invention can be prepared by methodswell known in the art. For example, such methods are described in thefollowing references: U.S. Pat. No. 5,041,449, PCT publication WO92/20669 (PCT application PCT/CA92/00211), Journal of Chromatography,645 (1993) 107-114, Tetrahedron Assymetry Vol. 4 No. 11 pp2319-2322(1993), Tetrahedron Assymetry Vol. 4 No. 2 pp211-214 (1993), Bioorganic& Medicinal Chemistry Vol.3 No.8, pp.1543-1546 (1993), TetrahedronLetters, Vol.33, No. 46, pp 6949-6952, (1992), J.Org. Chem., 34(6),1547-1550 (1969), J.Org. Chem., 52(9), 1794-1801 (1987), J.Am.Chem.Soc.,87(8), 1785-1788 (1965), J.Org. Chem. (1989), 54, 631-635. which are allincorporated by reference.

[0173] According to one embodiment, it will be appreciated that theamount of a compound of the present invention required for use intreatment will vary not only with the particular compound selected butalso with the route of administration, the nature of the condition forwhich treatment is required and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orveterinarian. In general however a suitable dose will be in the range offrom about 0.01 to about 750 mg/kg of body weight per day, preferably inthe range of 0.5 to 60 mg/kg/day, most preferably in the range of 1 to20 mg/kg/day.

[0174] The desired dose according to one embodiment is convenientlypresented in a single dose or as divided dose administered atappropriate intervals, for example as two, three, four or more doses perday.

[0175] In another embodiment, the compound is conveniently administeredin unit dosage form; for example containing 10 to 1500 mg, conveniently20 to 1000 mg, most conveniently 50 to 700 mg of active ingredient perunit dosage form.

[0176] According to another embodiment of the present invention, theactive ingredient is administered to achieve peak plasma concentrationsof the active compound of from about 1 to about 75 μM, preferably about2 to 50 μM, most preferably about 3 to about 30 μM. This may beachieved, for example, by the intravenous injection of a 0.1 to 5%solution of the active ingredient, optionally in saline, or orallyadministered as a bolus containing about 1 to about 500 mg of the activeingredient. Desirable blood levels may be maintained by a continuousinfusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.4 to about 15 mg/kg of theactive ingredient.

[0177] While it is possible that, for use in therapy, a compound of thepresent invention may be administered as the raw chemical, it ispreferable according to one embodiment of the invention, to present theactive ingredient as a pharmaceutical formulation The embodiment of theinvention thus further provides a pharmaceutical formulation comprisinga compound of formula (I),or formula (II), or a pharmaceuticallyacceptable salt thereof together with one or more pharmaceuticallyacceptable carriers therefor and, optionally, other therapeutic and/orprophylactic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

[0178] According to one embodiment of the present invention,pharmaceutical formulations include but are not limited to thosesuitable for oral, rectal, nasal, topical (including buccal andsub-lingual), transdermal, vaginal or parenteral (includingintramuscular, sub-cutaneous and intravenous) administration or in aform suitable for administration by inhalation or insufflation. Theformulations may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods according to this embodimentinclude the step of bringing into association the active compound withliquid carriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

[0179] According to another embodiment, pharmaceutical formulationsuitable for oral administration are conveniently presented as discreteunits such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient; as a powder or granules.In another embodiment, the formulation is presented as a solution, asuspension or as an emulsion. Still in another embodiment, the activeingredient is presented as a bolus, electuary or paste. Tablets andcapsules for oral administration may contain conventional excipientssuch as binding agents, fillers, lubricants, disintegrants, or wettingagents. The tablets may be coated according to methods well known in theart. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservatives.

[0180] The compounds of the present invention according to an embodimentare formulated for parenteral administration (e.g. by injection, forexample bolus injection or continuous infusion) and may be presented inunit dose form in ampoules, pre-filled syringes, small volume infusionor in multi-dose containers with an added preservative. The compositionsmay take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing an/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

[0181] For topical administration to the epidermis, the compounds,according to one embodiment of the present invention, are formulated asointments, creams or lotions, or as a transdermal patch. Suchtransdermal patches may contain penetration enhancers such as linalool,carvacrol, thymol, citral, menthol and t-anethole. Ointments and creamsmay, for example, be formulated with an aqueous or oily base with theaddition of suitable thickening and/or gelling agents. Lotions may beformulated with an aqueous or oily base and will in general also containone or more emulsifying agents, stabilizing agents, dispersing agents,suspending agents, thickening agents, or colouring agents.

[0182] Formulations suitable for topical administration in the mouthinclude lozenges comprising active ingredient in a flavoured base,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base such as gelatin and glycerin orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier.

[0183] Pharmaceutical formulations suitable for rectal administrationwherein the carrier is a solid. In another embodiment, they arepresented as unit dose suppositories. Suitable carriers include cocoabutter and other materials commonly used in the art, and thesuppositories may be conveniently formed by admixture of the activecompound with the softened or melted carrier(s) followed by chilling andshaping in moulds.

[0184] According to one embodiment, the formulations suitable forvaginal administration are presented as pessaries, tampons, creams,gels, pastes, foams or sprays containing in addition to the activeingredient such carriers as are known in the art to be appropriate.

[0185] For intra-nasal administration the compounds, in one embodimentof the invention, are used as a liquid spray or dispersible powder or inthe form of drops. Drops may be formulated with an aqueous ornon-aqueous base also comprising one more dispersing agents,solubilising agents or suspending agents. Liquid sprays are convenientlydelivered from pressurized packs.

[0186] For administration by inhalation the compounds, according to oneembodiment of the invention are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. In another embodiment, pressurized packscomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas.

[0187] In another embodiment, the dosage unit in the pressurized aerosolis determined by providing a valve to deliver a metered amount.

[0188] Alternatively, in another embodiment, for administration byinhalation or insufflation, the compounds according to the presentinvention are in the form of a dry powder composition, for example apowder mix of the compound and a suitable powder base such as lactose orstarch. In another embodiment, the powder composition is presented inunit dosage form in, for example, capsules or cartridges or e.g. gelatinor blister packs from which the powder may be administered with the aidof an inhalator or insufflator.

[0189] In one embodiment, the above described formulations are adaptedto give sustained release of the active ingredient.

[0190] The compounds of the invention may also be used in combinationwith other antiviral agents.

[0191] In one embodiment, the compounds of the invention may be employedtogether with at least one other antiviral agent chosen from proteaseinhibitors, polymerase inhibitors, and helicase inhibitors.

[0192] As used in this application, the term “interferon” include:

[0193] interferon likes molecules such as interferon (IFN), interferonα-2a, interferon α-2b, consensus interferon (CIFN) and other types ofinterferons.

[0194] In one embodiment , the compounds of the invention may beemployed together with at least one other antiviral agent chosen frominterferon (IFN), interferon α-2a, interferon α-2b, consensus interferon(CIFN), ribavirin, amantadine, rimantadine, interleukine-12,ursodeoxycholic acid (UDCA), glycyrrhizin and silybum marianum.

[0195] In one embodiment, the compounds of the invention may be employedtogether with at least one other antiviral agent chosen fromInterferon-α, Ribavirin and Amantadine.

[0196] In one embodiment, the compounds of the invention may be employedtogether with at least one other antiviral agent chosen fromInterferon-α and Ribavirin (REBETRON).

[0197] In one embodiment, the compounds of the invention may be employedtogether Interferon-α.

[0198] In one embodiment, the compounds of the invention may be employedtogether with Ribavirin.

[0199] The combinations referred to above may conveniently be presentedfor use in the form of a pharmaceutical formulation and thuspharmaceutical formulations comprising a combination as defined abovetogether with a pharmaceutically acceptable carrier therefor comprise afurther aspect of the invention.

[0200] The individual components of such combinations may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

[0201] When the compound (I), or (II) or a pharmaceutically acceptablesalts thereof is used in combination with a second therapeutic agentactive against the same virus the dose of each compound may be eitherthe same as or differ from that when the compound is used alone.Appropriate doses will be readily appreciated by those skilled in theart.

[0202] The following examples are provided to illustrate variousembodiments of the present invention and shall not be considered aslimiting in scope.

[0203] The target compound can be prepared according to the abovescheme:

[0204] Step a: 2-benzoyloxy-acetaldehyde I reacted with methyl (R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxylate II in the presencepara-toluene sulfonic acid (pTSA) under transketalisation to give2-benzoyloxymethyl-1,3-dioxolane-4-carboxylmethyl ester III as a mixtureof cis and trans isomers in a ratio of 3:1 in favor of cis isomer.

[0205] Step b: The carboxylic methyl ester III was selectivelyhydrolysed using lithium hydroxide to give the corresponding acidderivatives IVa and IVb. The mixture were separated by flashchromatography and each isomer was further used independently.

[0206] Step c: The carboxylic function of IVa was then converted to anacetoxy leaving group by treatment with lead tetraacetate.

[0207] Step d: The (2R)-2-benzoyloxymethyl-1,3-dioxolane-4-acetoxy IVawas coupled with silylated 2-amino-6-chloropurine using trimethylsilyltrifluoromethylsulfonate (TMSTf) as activator to give a mixture of cisand trans isomers of nucleoside analogues VIa and VIb in a ratio of1.2:1 in favor of cis isomer. The mixture was separated by flashchromatography and each isomer was used independently further.

[0208] Step e: The (−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolaneVIa was treated with cyclopropylamine in ethanol to give thecorresponding(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolaneVII in good yield.

[0209] Step f: Removal of benzoyl protecting group was achieved bytreatment of(−)-(2R,4R)-2-benzoylbxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolaneVII with methanolic ammonia to give the desired product(−)-(2R,4R)-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane1(-) in good yield.

EXAMPLE 1

[0210]Methyl-2-(R,S)-benzoyloxymethyl-1,3-dioxolane-4-(R)-carboxlate.(III)

[0211] To a solution of methyl-α,β-isopropylidene-D-glycerate (II)(Fluka: registry #52373-72-5), (9.76 g, 60.9 mmol, 1 eq) andbenzoyloxyacetaldehyde I (10 g, 60.9 mmol, 1 eq) in toluene (20 mL) at80° C., p-toluenesulfonic acid (PTSA) (460 mg, 2.4 mmol, 4 mol %) wasadded. The reaction flask was kept under vacuum for one hour and adistillate was collected (80-85° C.) during this period of time. Theresidue was then cooled to room temperature (RT) and purified by columnchromatography on silica gel, using hexanes/ethyl acetate as eluent toproduce 13.2 g (81%) of the title compound as a mixture of cis and transisomers in a ratio of 3:1.

[0212] Cis isomer:

[0213]¹H-NMR (CDCl₃): δ(ppm): 3.75 (s, 3H, CH₃); 4.15(dd, 1H, C₅—CH),4.30 (dd, 1H, C₅-CH) ; 4.5 (m, 2H, CH₂—O—CO—C₆H₅); 4.7 -m, 1H, C₄—CH);5.4 t, 1H, C₂—CH) ; 7.45-8.1 (m, 5H, Ar—CH).

[0214] Trans isomer:

[0215]¹H-NMR (CDCl₃): δ(ppm): 3.8 (s, 3H, CH₃); 4.1(dd, 1H, C₅—CH); 4.35(dd, 1H, C₅—CH) ; 4.45 (m, 2H, CH₂—O—CO—C₆H₅); 4.75 (m, 1H, C₄—CH); 5.5(t, 1H, C₂—CH); 7.45-8.1 (m, 5H, Ar—CH).

EXAMPLE 2

[0216] (2R, 4R)-2-benzoyloxymethyl-1,3-dioxolane-4-carboxylic acid.(IVa)

[0217] (2S, 4R)-2-benzoyloxymethyl-1,3-dioxolane-4-carboxylic acid.(IVb)

[0218] To a solution of methyl-2-(R,S)-benzoyloxymethyl-1,3-dioxolane-4-(R)-carboxylate (III), (411 g, 1.54 mmol, 1 eq., 2:1 mixture of cisand trans isomers) in a 1:1 mixture of tetrahydrofuran (THF) and water,lithium hydroxide (LiOH) (64.8 g, 1.54 moles, 1 eq) was added portionwise over a period of 30 min., keeping the reaction flask temperaturebelow 30° C. After 90 min., THF was ;removed by vacuum and the aqueoussolution was acidified to pH=2.5-3.2, by dropwise addition of 30% (w/w)sulphuric acid. The resulting solution was extracted withdichloromethane (4×400 mL). The combined organic, phase was washed withbrine,, dried over sodium sulphate and concentrated to produce 380 g ofa dark oil. The isomers were separated by column chromatography onsilica gel, using 2% acetic acid in dichloromethane to produce 220 g ofthe cis isomer (IVa) (56.5%) and 116 g of the trans isomer (IVb) (30%).Each of isomers was independently used for next step.

[0219] Cis isomer:

[0220] (2R, 4R)-2-benzoyloxymethyl-1,3-dioxolane-4-carboxylic acid.(IVa)

[0221]¹H-NMR (CDCl₃): δ(ppm): 4.2 (t, 1H, C₅—H); 4.4 (m, 1H); 4.5 (m,1H); 4.7 (m, 2H); 5.4 (t, 1H, C₂—CH); 7.45-8.1 (m, 5H, Ar—CH);7.2-8.0(bs, 1H, COOH).

[0222] Trans isomer:

[0223] (2S, 4R)-2-benzoyloxymethyl-1,3-dioxolane-4-carboxylic acid.(IVb)

[0224]¹H-NMR (CDCl₃): δ(ppm): 4.15 (dd, 1H, C₅—H); 4.4 (t, 1H, C₅—H);4.45 (m, 2H, CH₂—OCOC₆H₅); 4.8 (dd, 1H, C₄—CH); 5.6 (t, 1H, C₂—CH);7.45-8.1 (m, 5H, Ar—CH); 8.3-8.8 (bs, 1H, COOH).

EXAMPLE 3

[0225] (2R)-2-benzoyloxymethyl-4-(R,S)-acetoxy-1,3-dioxolane.(V)

[0226] To a solution of(2R,4R)-2-benzoyloxymethyl-1,3-dioxolane-4-carboxylic acid (IVa), (130g, 0.515moles, 1 eq) and pyridine (60 mL, 0.741 moles, 1.44 eq) inacetonitrile at 4° C., lead tetraacetate. (assay 95%, 300 g, 0.678moles, 1.25 eq) was added over a period of 20 min. The reaction mixturewas kept under stirring for 18 hours at room temperature (RT). Theinorganics were removed by filtration, the filtrate was poured on to asaturated solution of sodium bicarbonate (2L) followed by addition ofsolid sodium bicarbonate (pH=7-8). The organic phase was separated, andthe aqueous phase was extracted with ethyl acetate (3×400 mL). Thecombined organic phase was concentrated and purified by columnchromatography, on silica gel, using hexanes/ethyl acetate as eluent toproduce 93.5 g (68%) of the title compound as a mixture of cis and transisomers in a ratio of 2:1 (V). The mixture was used for next step.cis/trans isomers:

[0227]¹H-NMR (CDCl₃): δ(ppm): 2.0,2.15 (s, 3H, CH₃); 4.05-4.45 (m, 4H,CH); 5.45, 5.55 (t, 1H, C₂—CH); 6.4, 6.45 (dd, 1H, C₄—CH); 7.45-8.1 (m,5H, Ar—CH);

EXAMPLE 4

[0228] (2R,4R) and(2R,4S)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIa and VIb)

[0229] 2-amino-6-chloro-purine (4.15 g, 1.3 eq.) in 50 ml ofhexamethyldisilazane(HMDS) containing 100 mg of ammonium sulfate washeated under reflux for 3 h after which time the clear solution wasevaporated to dryness in vacuo. The residue was dissolved in 100 mL ofanhydrous 1,2-dichloroethane.(2R)-2-benzoyloxymethyl-4-acetoxy-1,3-dioxolane (V) (5 g) was dried byco-evaporation twice with benzene (2×30 mL) and dissolved in 100 mL ofanhydrous 1,2-dichloroethane. The solution was then transferred into thereaction flask containing silylated 2-amino-6-chloro-purine solution.The mixture was placed in a 60° C. preheated oil bath for 15 minutes,followed the addition of trimethylsilyl triflate (TMS-OTf) (3.8 mL, 1.1eq.). The mixture was heated at refluxing under nitrogen for 3 h and thesolution became brown. TLC (hex:EtOAc 7:3 for sugar and hex:EtOAc 1:4for product) indicated a completed reaction with the disappearance ofsugar and the presence of two well separated spots for cis and transproducts. The reaction mixture was cooled to room temperature, pouredinto a saturated sodium bicarbonate solution (100 mL) and stirred for 10minutes. The organic layer was collected and the aqueous layer wasextracted twice with methylene chloride (2×50 ml). The combined organicsolution was washed with water, brine and dried over MgSO₄ as usual andsolvent was evaporated to dryness to give a foam (7 g). H-NMR of thecrude indicated clean reaction with cis and trans products in a ratio of1.2:1 in favor of cis isomer. The crude product was purified on silicagel using a gradient of hexane:ethyl acetate 7:3, 1:1 and 2:3 as eluantto yield 2.5 g of trans isomer (less polar, α-anomer) (VIb) as a foam,which was crystallized in EtOH and 3 g of cis isomer (more polar,β-anomer) (VIa) as a foam, which was crystallized in EtOH and 0.3 g ofmixture cis and trans in favor of cis as a foam for a total of 82%yield.

[0230] Trans isomer

[0231](+)-(2R,4S)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIb)

[0232] R_(f): 0.40 (hexane-EtOAc 3:7)

[0233] [α_(D)] +21.16° (c 0.293 in CH₂Cl₂)

[0234]¹H-NMR (CDCl₃): δ(ppm): 4.45-4.55 (m, 4H; C₅—H₂, C₂—CH₂—OBz), 5.16(b, 2H, NH₂ ), 5.83(t, 1H, C₂—H, J=3.8Hz), 6.39 (dd, 1H, C₄—H), 7.45 (t,2H, aromatic), 7.62 (t, 1H, aromatic), 7.92 (s, 1H, C_(8′)—H), 8.10 (d,2H, aromatic)

[0235] U.V.: (CH₃OH) λ_(max): 312 nM

[0236] Cis isomer

[0237](−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIa)

[0238] R_(f): 0.26 (hexane-EtOAc 3:7)

[0239] [α_(D)] 87.70° (c 0.2565 in CH₂Cl₂)

[0240]¹H-NMR (CDCl₃): δ(ppm): 4.25-4.33 (dd, 1H, C₅—H), 4.60-4.64 (m,3H; C₅—H and C₂—CH₂—OBz) , 5.17 (b, 2H, NH₂ ), 5.42 (t, 1H, C₂—H,J=3.5Hz) , 6.33 (dd, 1H, C₄—H), 7.45 (t, 2H, aromatic), 7.62 (t, 1H,aromatic), 7.95 (d, 2H, aromatic), 8.05 (s, 1H, C_(8′)—H).

[0241] U.V.: (CH₃OH) λ_(max): 312 nM.

EXAMPLE 5

[0242](−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane(VII)

[0243] To a solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIa) (600 mg) in ethanol (30 mL) was added cyclopropylamine (2 mL, =18eq.). The mixture was gently heated at reflux (80-85° C.) for 18 h andcooled to room temperature. Solvent was evaporated to dryness in vacuo.The residue was dissolved in 100 mL of methylene chloride, washed withsaturated NaHCO₃ solution, water, brine and dried over MgSO₄. Solventwas removed in vacuo and residue was purified on silica gel usingEtOAc:MeOH as eluant to give the desired product (VII) as a foam in 80%yield.(506 mg).

[0244] R_(f): 0.26 (CH₂Cl₂: MeOH 95:5)

[0245] [α_(D)] −67.70 (c 0.2565 in CH₂Cl₂)

[0246]¹H-NMR (CDCl₃): δ(ppm): 0.64-0.68 (m, 2H, CH₂ of cyclopropyl),0.91-0.96 (m, 2H, CH₂ of cyclopropyl), 3.06 (b, 1H, CH of cyclopropyl),4.27-4.30 ( dd, 1H, C₅—H), 4.54-4.57 -(dd, 1H ; C₅—H) 4.60 (t, 2H,C₂—CH₂—OBz), 5.37 (b, 2H, NH₂ ), 5.42 (t, 1H, C₂—H, J=3.5Hz), 6.28 (b,1H, NH) 6.35 (dd, 1H, C₄—H) 7.45 (t, 2H, aromatic), 7.58 (t, 1H,aromatic), 7.77 (s, 1H, C₈-8), 8.01(d, 2H, aromatic),

[0247] U.V.: (CH₃OH) λ_(max): 283 and 260 nM.

EXAMPLE 6

[0248](−)-(2R,4R)-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane(compound 1(−))

[0249] A solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane(VII) (480 mg) in 30 mL of saturated methanolic ammonia was stirred atroom temperature for 18 h. The mixture was evaporated to dryness invacuo. The residue was dissolved in 20 mL of water, washed twice with 10mL of methylene chloride and lyophilized to give 283 mg of white solidin 80% yield (1(−)).

[0250] R_(f): 0.26 (CH₂Cl₂: MeOH 9:1)

[0251] [α_(D)] −35.9° (c 0.334 in MeOH)

[0252]¹H-NMR (DMSO_(d-6)): δ(ppm): 0.55 (m, 2H, CH₂ of cyclopropyl),0.95 (m, 2H, CH₂ of cyclopropyl), 3.15 (b, 1H, CH of cyclopropyl), 3.80(m, 2H, CH₂OH) , 4.30 ( dd, 1H, C₅—H), 4.55 (dd, 1H ; C₅—H), 5.08 (t,1H, C₂OH), 5.17 (b, H, OH) , 6.15 (b, 2H, NH₂), 6.52 (dd, 1H, C₄—H),7.72 (b, 1H, NH), 8.12 (s, 1H, C_(8′) -8).

[0253] U.V. (CH₃OH) λ_(max): 283 and 260 nM.

EXAMPLE 7

[0254](−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane

[0255] To a solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIa) (250 mg) in ethanol (25 ml) was added cyclobutylamine (0.17 mL, =3eq.). The mixture was gently heated at reflux (80-85° C.) for 18 h andcooled to room temperature. Solvent was evaporated to dryness in vacuo.The residue was dissolved in 100 mL of methylene chloride, washed withsaturated NaHCO₃ solution, water, brine and dried over MgSO₄. Solventwas removed in vacuo and residue was purified on silica gel usingEtOAc:MeOH 95:5 as eluant to give the desired product as a foam in 84%yield.(230 mg).

[0256] R_(f): 0.31 (CH₂Cl₂: MeOH 95:5)

[0257] [α_(D)] −62.5° (c 0.4925 in CH₂Cl₂)

[0258]¹H-NMR (CDCl₃): δ(ppm): 1.74-1.78 (m, 2H, CH₂ of cyclobuyl),1.95-2.00 (m, 2H, CH₂ of cyclobutyl), 2.43-2.45 (m, 2H, CH₂ ofcyclobutyl), 4.27-4.30 ( dd, 1H, C₅-H), 4.54-4.57 (dd, 1H; C₅—H), 4.59(t, 2H, C₂—CH₂—OBz), 4.75 (b, 1H, CH of cyclobutyl), 5.37 (b, 2H, NH₂),5.41 (t, 1H, C₂—H, J=3.6Hz), 6.00 (b, 1H, NH), 6.35 (dd, 1H, C₄—H), 7.45(t, 2H, aromatic), 7.58 (t, 1H, aromatic), 7.75 (s, 1H, C_(8′)—H),8.01(d, 2H, aromatic),

[0259] U.V.: (CH₃OH) λ_(max): 283 and 263 nM.

EXAMPLE 8

[0260] (−) -(2R,4R)-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane(compound 2(−))

[0261] A solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane(214 mg) in 20 mL of saturated methanolic ammonia was stirred at roomtemperature for 18 h. The mixture was evaporated to dryness in vacuo.The residue was dissolved in 20 mL of water, washed twice with 10 mL ofether and evaporated to dryness by coevaporation with ethanol to give154 mg of pure product (2(−)) as a foam in 96% yield.

[0262] R_(f): 0.52 (CH₂Cl₂:MeOH 9:1)

[0263] [α_(D)] −29.04° (c 0.396 in MeOH)

[0264]¹H-NMR (DMSO_(d-6)): δ(ppm): 1.61 (m, 2H, CH₂ of cyclobutyl), 2.06(m, 2H, CH₂ of cyclobutyl) , 2.18 (m, 2H, CH₂ of cyclobutyl), 3.58 (m,2H, CH₂OH) , 4.17 ( dd, 1H, C₄—H), 4.40 (dd, 1H ; C₅—H), 4.90 (b, 1H, CHof cyclobutyl), 5.01 (t, 1H, C₂—H), 5.42 (b, H, OH), 5.87 (b, 2H, NH₂ ),6.19 (dd, 1H, C₄—H) 7.62 (b, 1H, NH), 7.85 (s, 1H, C_(8′)-8).

[0265] U.V.: (CH₃₀H) λ_(max): 283 and 260 nM.

EXAMPLE 9

[0266](−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane

[0267] To a solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-chloro-purine-9′-yl)-1,3-dioxolane(VIa) (250 mg) in ethanol (15 mL) was added cyclopentylamine (0.2 mL, =3eq.). The mixture was gently heated at reflux (80-85° C.) for 18 h andcooled to room temperature. Solvent was evaporated to dryness in vacuo.The residue was dissolved in 100 mL of methylene chloride, washed withsaturated NaHCO₃ solution, water, brine and dried over MgSO₄. Solventwas removed in vacuo and residue was purified on silica gel using EtOACand EtOAc:MeOH 95:5 as eluant to give the desired product as a foam in70% yield.(191 mg).

[0268] R_(f): 0.30 (CH₂Cl₂ : MeOH 95:5)

[0269] [α_(D)] −67.7° (c 0.363 in CH₂Cl₂)

[0270]¹H-NMR (CDCl₃): δ(ppm): 1.53 (m, 2H, CH₂ of cyclopentyl), 1.68 (m,2H, CH₂ of cyclopentyl), 1.76 (m, 2H, CH₂ of cyclopentyl), 2.10 (m, 2H,CH₂ of cyclopentyl), 4.25 ( dd, 1H, C₅—H), 4.54-4.54-4.60 (m, 4H; C₅—H,C₂—CH₂—OBz and CH of cyclopentyl), 4.98 (b, 2H, NH₂ ), 5.42 (t, 1H,C₂—H, J=3.5Hz) , 5.75 (b, 1H, NH), 6.35 (d, 1H, C₄—H), 7.45 (t, 2H,aromatic), 7.58 (t, 1H, aromatic), 7.74 (s, 1H, C_(8′)—H), 8.01(d, 2H,aromatic),

[0271] U.V.: (CH₃OH) ) λ_(max): 283 and 260 nM.

EXAMPLE 10

[0272](−))-(2R,4R)-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane(compound 3(−))

[0273] A solution of(−)-(2R,4R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane(180 mg) in 20 mL of saturated methanolic ammonia was stirred at roomtemperature for 18 h. The mixture was evaporated to dryness in vacuo.The residue was purified on silica gel using CH₂Cl₂: MeOH 95:5 as eluantto give 130 mg of white solid (3(−))in 95% yield.

[0274] R_(f): 0.58 (CH₂Cl₂: MeOH 9:1)

[0275] [α_(D)] −30.2° (c 0.4275 in MeOH)

[0276]¹H-NMR (DMSO_(d-6)): δ(ppm): 1.55 (m, 4H, CH₂ of cyclopentyl),1.68 (m, 2H, CH₂ of cyclopentyl), 1.88 (m, 2H, CH₂ of cyclopentyl), 3.58(m, 2H, CH₂OH), 4.17 ( dd, 1H, C₅—H) , 4.41 (dd, 1H ; C₅—H), 4.5 (b, 1H,CH of cyclopentyl), 5.01 (t, 1H, C₂—H), 5.14 (b, H, OH), 5.8-5 (b, 2H,NH₂ ), 6.18 (dd, 1H, C₄-H), 7.12 (b, 1H, NH), 7.84 (s, 1H, C_(8′)—H).

[0277] U.V. : (CH₃OH) λ_(max):283 and 260 nm.

EXAMPLE 11

[0278] Preparation of(+)-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane (β-D-OddC)(compound#17(+)) and(+)-triphosphate-cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane(β-D-OddC-tp)(compounds #22(+))

[0279] To a stirring suspension of4-amino-1-(2-(R)-hydroxymethyl-[1,3]dioxolan-4-(R)-yl)-1H-pyrimidin-2-one(17(+)) (18.8 mg, 0.088 mmol) in dry-DMF (0.49 mL) was added drypyridine (0.13 mL) followed by a freshly prepared solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one 1.0 M in 1,4-dioxane (81 μL, 0.081mmol). The mixture was stirred 15 minutes at room temperature, thentributylamine (53 μL, 0.22 mmol) and a solution of tributylammoniumpyrophosphate 0.42 M in DMF (175 μL, 0.073 mmol) were addedsimultaneously. The mixture was stirred another 15 minutes. A solutionof I2 1% in pyridine/H2O (98:2) (1.4 mL, 0.115 mmol of I) was added andthe mixture was stirred 15 minutes. The excess of iodine was destroyedby adding 0.5 mL of aqueous sodium bisulfite 5%. The mixture was stirred15 minutes, then it was concentrated under reduced pressure to removeall solvents. The residue was dissolved in water, washed two times withmethylene chloride and once with ethyl acetate. The aqueous layer wasconcentrated and purified by charcoal column as follow: about 400 mg ofcharcoal, placed over a thin layer of Celite in a funnel with fritteddisk, was prewashed by passing methanol, then water (by vaccuum). Thecrude residue was diluted in a minimum of water, acidified to pH 1-2 byadding few drops of HCl 1N, then placed on the top of the charcoalcolumn. The column was eluted with water (25 mL) in order to removeinorganic salts, then 0.5 N ammonia (10 mL) to collect the desiredtriphosphate. The collected triphophate was lyophilized, and it was thenpurified again on a small pad of charcoal, this time eluting only withwater. The desired triphosphate comes out fast. Few fractions werecollected and lyophilized to give the triphosphate ammonium salt(22(+)), as a yellow solid (4.8 mg, 13% yield).

[0280]¹H NMR (400 MHz, D₂O) δ ppm: 7.93 (d, 1 H, 7.47 Hz), 6.21 (s, 1H), 6.02 (d, 1 H, 7.47 Hz), 5.19 (s, 1 H), 4.20 (m, 4 H).

[0281]³¹P NMR (162 MHz, D₂O) δ ppm: −6.1 (d, 19 Hz), −10.5 (d, 19 Hz),−21.7 (t, 19 Hz).

[0282] The compounds of the present invention can be prepared by methodswell known in the art. For example, such methods are described in thefollowing references: U.S. Pat. No. 5,041,449, PCT publication WO92/20669 (PCT application PCT/CA92/00211), Journal of Chromatography,645 (1993) 107-114, Tetrahedron Assymetry Vol. 4 No. 11 pp.2319-2322(1993), Tetrahedron Assymetry Vol. 4 No. 2 pp.211-214 (1993), Bioorganic& Medicinal Chemistry Vol.3 No.8, pp.1543-1546 (1993), TetrahedronLetters, Vol.33, No. 46, pp 6949-6952, (1992), J.Org. Chem., 34(6),pp.1547-1550 (1969), J.Org. Chem., 52(9), pp.1794-1801 (1987),J.Am.Chem.Soc., 87(8), pp.1785-1788 (1965), J.Org. Chem. (1989), 54,pp.631-635 which are all incorporated by reference.

[0283] In a similar manner, the following compounds were synthesized:

[0284] (−)Cis-2-hydroxymethyl-4-(5′-cytosin-1′-yl)-1,3-Dioxolane(Compound #17(−))

[0285] (+)Cis-2-hydroxymethyl-4-(5′-cytosin-1′-yl)-1,3-Dioxolane(Compound #17 (+))

[0286] (+)Cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-Dioxolane(Compound#18 (+))

[0287] (+)Cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-Dioxolane(Compound#18 (−))

[0288] (+)Cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-Dioxolane(Compound#19 (+))

[0289] (−)Cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-Dioxolane(Compound#19 (−))

[0290] (−)-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(β-L-) (compound#20 (−)) .

[0291] (+)-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(compound#20 (+))

[0292] (−)-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane(compound#21 (−))

[0293] (+)-cis-2-hydroxymethyl-4-(N-1′-thyminyl)-1,3-dioxolane(compound#21 (+))

EXAMPLE 12 Evaluation of Nucleoside Triphosphate Analogues In The HCVRNA-Dependent RNA Polymerase Assay

[0294] The following references which are referenced in the example areall incorporated by reference:

[0295] 1. Behrens, S., Tomei, L., De Francesco, R. (1996) EMBO 15,pp.12-22

[0296] 2. Harlow, E, and Lane, D. (1988) Antibodies: A LaboratoryManual. Cold Spring Harbord Laboratory. Cold Spring Harbord. NY.

[0297] 3. Lohmann, V., Körner, F., Herian, U., and Bartenschlager, R.(1997) J. Virol. 71, pp.8416-8428

[0298] Compounds were evaluated using an in vitro polymerase assaycontaining purified recombinant HCV RNA-dependent RNA polymerase (NS5Bprotein). HCV NS5B was expressed in insect cells using a recombinantbaculovirus as vector. The experimental procedures used for the cloning,expression and purification of the HCV NS5B protein are describedbellow. Follows, are details of the RNA-dependent RNA polymerase assaysused to test the compounds.

[0299] Expression of the HCV NS5B Protein in Insect Cells:

[0300] The cDNA encoding the entire NS5B protein of HCV-Bk strain,genotype 1b, was amplified by PCR using a plasmid containing a cDNAversion of the full-length HCV genome as template. The oligonucleotidesused to amplify this HCV region were designed to introduce a NheI sitefollowed by an ATG at the 5′ end of the NS5B coding region as well as aBamHI site at the 3′end immediately downstream of the translation stopcodon. The amplified sequence, of 1.8 kb, was digested with NheI andBamHI and ligated to a predigested pBlueBacII plasmid (Invitrogen). Theresulting recombinant plasmid was designated pBac/NS5B. Sf9 cells wereco-transfected with 3 μg of pBac/NS5B, together with 1 μg of linearizedbaculovirus DNA (Invitrogen), as described in the manufacturer'sprotocol. Following two rounds of plaque purification, anNS5B-recombinant baculovirus, BacNS5B, was isolated. The presence of therecombinant NS5B protein was determined by western blot analysis (Harlowand Lane, 1988) of BacNS5B-infected Sf9 cells, using a HCV NS5B specificrabbit polyclonal antiserum (anti-NS5B). Infections of Sf9 cells withthis plaque -purified virus were performed in one-liter spinner flasksat a cell density of 1.2×10⁶ cells/ml and a multiplicity of infection of5.

[0301] Preparation of a Soluble Recombinant NS5B Protein:

[0302] Sf9 cells were infected as described above. Sixty hourspost-infection, cells were harvested then washed twice with phosphatebuffer saline (PBS). Total proteins were solubilized as described inLohmann et al. (1997) with some modifications. In brief, proteins wereextracted in three steps, S1, S2, S3, using lysis buffers (LB) I, LB IIand LB III (Lohmann et al, 1997). The composition of LBII was modifiedto contain 0.1% triton X-100 and 150 mM NaCl to reduce the amount ofsolubilized NS5B protein at this step. In addition, sonication of cellextracts was avoided throughout the protocol to preserve the integrityof the protein structure.

[0303] Purification of recombinant NS5B using fast protein liquidchromatography (FPLC):

[0304] Soluble NS5B protein in the S3 fraction was diluted to lower theNaCl concentration to 300 mM, then it incubated batchwise with DEAEsepharose beads (Amersham-Pharmacia) for 2 hrs at 4° C., as described byBehrens et al. (1996). Unbound material was cleared by centrifugationfor 15 min at 4° C., at 25 000 rpm using a SW41 rotor (Beckman). Thesupernatant was further diluted to lower the NaCl concentration to 200mM and subsequently loaded, with a flow rate of 1 ml/min, on a 5 mlHiTrap® heparin column (Amersham-Pharmacia) connected to an FPLCO system(Amersham-Pharmacia). Bound proteins were eluted in 1 ml fractions,using a continuous NaCl gradient of 0.2 to 1 M, over a 25 ml volume.NS5B-containing fractions were identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by westernblotting using the anti-NS5B antiserum at a dilution of 1:2000. Positivefractions were pooled and the elution buffer was exchanged against a 50mM NaPO₄ pH 7.0, 20% glycerol, 0.5% triton X-100 and 10 mM DTT, using aPD-10 column (Amersham-Pharmacia). The sample was then loaded onto a 1ml HiTrap® SP column (Amersham-Pharmacia), with a flow rate of 0.1ml/min. Bound proteins were eluted using a continuous 0 to 1 M NaClgradient over a 15 ml volume. Eluted fractions were analyzed by SDS-PAGEand western blotting. Alternatively, proteins were visualized, followingSDS-PAGE, by silver staining using the Silver Stain Plus kit (BioRad) asdescribed by the manufacturer. Positive fractions were tested for RdRpactivity (see below) and the most active ones were pooled, and stored asa 40% glycerol solution at −70° C.

[0305] In Vitro RNA-dependent RNA Polymerase Assays Used to EvaluateTriphosphate Form of Guanosine Analogues:

[0306] RdRp assays were conducted using either homopolymerictemplate/primers or in vitro transcribed RNA templates.

[0307] All RdRp reactions which used homopolymeric template/primers wereperformed in a total volume of 50 μl of a buffer consisting of 20 mMTris-HCl pH 7.5, 1 mM DTT, 50 mM NaCl, 0.5 mM MnCl₂, 5 μCi [γ ³²P]-GTP(3000 Ci/mmol), and 4 μM of cold GTP. Standard HCV RdRp reactionscontained 200 ng of purified NS5B protein. Guanosine analogues weretested in the presence of polyC/oligoG. PolyC RNAs (Amersham-Pharmacia)was resuspended at 400 ng/μl. The primer oligoG₁₂ (MWG, Germany) wasdiluted to a concentration of 20 pmol/μl (7.6 ng/μl). Templates andprimers were mixed volume to volume, denatured at 95° C. for 5 min andannealed at 37° C. for 10 min. Following a two hour incubation at 22°C., reactions were stopped by the addition of 100 μg of sonicated salmonsperm DNA (Life Technologies) and 1 ml of 10% trichloroacetic acid-0.5%tetrasodium pyrophosphate (TCA-PPi). Nucleic acids were precipitated at4° C. for 30 min after which samples were filtered on GF/C glassmicrofiber filters (Millipore). Membranes were subsequently washed with25 ml of a 1% TCA-0.1% PPi solution, then air dried. Incorporatedradioactivity was quantified using a liquid scintillation counter(1450-Microbeta, Wallac).

[0308] Heteropolymeric RNA templates were generated by run-offtranscription. As template for these transcription reactions, arecombinant pcDNA3 plasmid (Invitrogen) containing a cDNA version of theHCV genome was used and referred to as pcDNA/HCVfl. In vitrotranscriptions were performed using the MEGAscript™ kit (Ambion), assuggested by the manufacturer. In brief, the plasmid pcDNA/HCVfl waslinearized with EcoRI to generate a truncated HCV transcript of about6900 nucleotides. Linearized DNA was extracted with a one to one volumeof phenol/chloroform, precipitated with ethanol, then 1 μg of thislinearized DNA was used as template in T7 RNA polymerase-driven in vitrotranscription reactions. Transcripts were extracted using the TRIZOL®reagent (Life Technologies) and an aliquot (1 μg) was used as templatein RdRp assays. RdRp reactions using heteropolymeric templates werecarried out under the same conditions as described above forhomopolymeric template/primers, with the exception that the substratemixture composition consisted of 5 μCi of [γ ³²P]-GTP, 1 μM cold GTP and100 μM of the remaining nucleotides (ATP, CTP and UTP). Reactionmixtures were subsequently processed as described above forhomopolymeric template-containing reactions. Results are shown below:HCV polymerase Compound % inhibition 9(−)-triphosphate 44% @ 100 μM13(−) triphosphate 82% @ 50 μM IC₅₀ = 10 μM 14(−)-triphosphate 6% @ 50μM

[0309] In Vitro RNA-dependent RNA Polymerase Assays Used to EvaluateTriphosphate Form of Cytosine Analogues:

[0310] RdRp assays were conducted using either homopolymerictemplate/primers or in vitro transcribed RNA templates.

[0311] All RdRp reactions which used homopolymeric template/primers wereperformed in a total volume of 50 μl of a buffer consisting of 20 mMTris-HCl pH 7.5, 1 mM DTT, 50 mM NaCl, 0.5 mM MnCl₂, 5 μCi [γ ³²P]-CTP(3000 Ci/mmol), and 5 μM of cold CTP. Standard HCV RdRp reactionscontained 200 ng of purified NS5B protein. Cytosine analogues weretested in the presence of polyrI/oligodC. PolyrI RNAs(Amersham-Pharmacia) was resuspended at 400 ng/μl. The primer oligodC₁₂(Life Technologies) was diluted to a concentration of 20 pmol/μl.Templates and primers were mixed volume to volume, denatured at 95° C.for 5 min and annealed at 37° C. for 10 min. Following a two hourincubation at 22° C., reactions were stopped by the addition of 100 μgof sonicated salmon sperm DNA (Life Technologies) and 1 ml of 10%trichloroacetic acid-0.5% tetrasodium pyrophosphate (TCA-PPi). Nucleicacids were precipitated at 4° C. for 30 min after which samples werefiltered on GF/C glass microfiber filters (Millipore). Membranes weresubsequently washed with 25 ml of a 1% TCA-0.1% PPi solution, then airdried. Incorporated radioactivity was quantified using a liquidscintillation counter (1450-Microbeta, Wallac).

[0312] Heteropolymeric RNA templates were generated by run-offtranscription. As template for these transcription reactions, arecombinant pcDNA3 plasmid (Invitrogen) containing a cDNA version of theHCV genome was used and referred to as pcDNA/HCVfl. In vitrotranscriptions were performed using the MEGAscript™ kit (Ambion), assuggested by the manufacturer. In brief, the plasmid pcDNA/HCVfl waslinearized with EcoRI to generate a truncated HCV transcript of about6900 nucleotides. Linearized DNA was extracted with a one to one volumeof phenol/chloroform, precipitated with ethanol, then 1 μg of thislinearized DNA was used as template in T7 RNA polymerase-driven in vitrotranscription reactions. Transcripts were extracted using the TRIZOL®reagent (Life Technologies) and an aliquot (1 μg) was used as templatein RdRp assays.

[0313] RdRp reactions using heteropolymeric templates were carried outunder the same conditions as described above for homopolymerictemplate/primers, with the exception that the substrate mixturecomposition consisted of 5 μCi of [γ ³²P]-CTP, 1 μM cold CTP and 100 μMof the remaining nucleotides (ATP, GTP and UTP). Reaction mixtures weresubsequently processed as described above for homopolymerictemplate-containing reactions. Results are shown below: HCV polymeraseCOMPOUND % inhibition COMPOUND#22(+) 85% @ 50 μM IC₅₀ = 7.7 μMCOMPOUND#22(−) 15% @ 100 μM COMPOUND#23(+) 57% @ 100 μM COMPOUND#23(−)81% @ 100 μM IC₅₀ = 16 μM COMPOUND#24(+) 12% @ 50 μM COMPOUND#24(−) 8% @100 μM COMPOUND#25(+) 9% @ 50 μM COMPOUND#26(+) 47% @ 100 μM

EXAMPLE 13 Cytotoxicity Assay

[0314] The cytotoxicity of test compounds was evaluated according to thefollowing procedure:

[0315] Flat bottom 96 well plates were plated with 5X10E3 Vero-34cells/well and 1X10E4 Hs-68 or Wi-38 cells/well respectively andincubated overnight at 37° C. and 5% CO₂/air. After incubation, thesupernatant medium was removed and replaced with test compound dilutionsin 2% DMEM (150 μl). The cells were then incubated 48 hours in a 5% CO₂incubator at 37° C.

[0316] 50 μl/well of 10 μCi/ml solution of [³H]-methyl thymidine(specific activity of approx. 2Ci/mmol) was added to the culture mediumand incubated overnight (18 hours) in a 5% CO₂ incubator at 37° C.

[0317] Cells were then collected onto a fiberglass filter (PrintedFiltermat A 1450-421 Wallac) with a Tomtec cell harvester. Suspendedcells were collected directly onto filter while for adherent cells, themedium was first removed, then the cells washed with PBS and trypsinizedfor 2-3 minutes (50 μl trypsin/well) before collecting.

[0318] Filters were dried for 1 hour at 37-40° C. and then placed intobags (1450-microbeta # 1450-432 Wallac) with 4.5 ml of Betascint andcounts obtained with Microbeta 1450 Wallac.

[0319] The percent of cell proliferation was determined by comparison tothe control (no test compound) and thereby establishing 50% inhibitoryconcentration is established.

We claim:
 1. A method for treating or preventing a Flaviviridae viralinfection in a host comprising administering a therapeutically effectiveamount of at least one compound having the formula I or apharmaceutically acceptable salt thereof:

wherein: R is H, —NR₂R₃ or OR₄ wherein R₂ is H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₈cycloalkyl; R₃ is H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl; R₄ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl;and Ra is chosen from H, monophosphate, diphosphate, triphosphate,carbonyl substituted with a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₆₋₁₀ aryl and

wherein each Rc is independently chosen from H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and an hydroxy protecting group; wherein saidnucleoside is present in the form of the (−) enantiomer, (+) enantiomerand mixtures thereof, including racemic mixtures.
 2. The method of claim1 wherein R is NH₂, H or OH.
 3. The method of claim 1 wherein R is NH₂or OH.
 4. The method of claim 1 wherein R is OH.
 5. The method of claim1 wherein R is —NR₂R₃ wherein R₂ is cycloropyl and R₃ is H.
 6. Themethod of claim 1 wherein Ra is chosen from H, monophosphate,diphosphate, and triphosphate.
 7. The method of claim 1 wherein Ra ischosen from monophosphate, diphosphate, and triphosphate.
 8. The methodof claim 1 wherein Ra is triphosphate.
 9. The method of claim 1 whereinRa is H.
 10. The method as defined in claim 1 wherein said compound offormula I of the present invention is substantially in the form of the(−) enantiomer.
 11. The method of claim 1 which comprises administeringat least one compound chosen from: Compound 1cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;Compound 2cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane;Compound 3cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 4-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 5 cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane; Compound6 cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane; Compound 7cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane; orCompound 8 cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane.12. The method of claim 1 which comprises administering at least onecompound chosen from Compound 1(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane;Compound 2(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclobutylamino-purine-9′-yl)-1,3-dioxolane;Compound 3(−)cis-2-hydroxymethyl-4-(2′-amino-6′-cyclopentylamino-purine-9′-yl)-1,3-dioxolane;Compound 4(−)-cis-2-hydroxymethyl-4-(2′-6′-diamino-purin-9′-yl)-1,3-dioxolane;Compound 5(−) cis-2-hydroxymethyl-4-(guanin-9′-yl)-1,3-dioxolane;Compound 6) (−) cis-2-hydroxymethyl-4-(adenin-9′-yl)-1,3-dioxolane;Compound 7(−)cis-2-hydroxymethyl-4-(2′amino-6′-chloro-purin-9′-yl)-1,3-dioxolane; orCompound 8(−) cis-2-hydroxymethyl-4-(2′amino-purin-9′-yl)-1,3-dioxolane.13. The method as defined in claim 1 wherein said Flaviviridae viralinfection is hepatitis C (HCV).
 14. The method as defined in claim 10wherein said Flaviviridae viral infection is hepatitis C (HCV).
 15. Themethod as defined in claim 11 wherein said Flaviviridae viral infectionis hepatitis C (HCV).
 16. The method as defined in claim 12 wherein saidFlaviviridae viral infection is hepatitis C (HCV).
 17. A method fortreating or preventing a hepatitis C viral infection in a hostcomprising administering to the host a therapeutically effective amountof at least one compound according to formula I as defined in claim 1and at least one further antiviral agent.
 18. The method according toclaim 17 wherein the antiviral agent is chosen from viral serineprotease inhibitor, viral polymerase inhibitor and viral helicaseinhibitor.
 19. The method according to claim 17 wherein the antiviralagent chosen from interferon (IFN), interferon α-2a, interferon α-2b,consensus interferon (CIFN), ribavirin, amantadine, rimantadine,interleukine-12, ursodeoxycholic acid (UDCA), glycyrrhizin and silybummarianum.
 20. The method according to claim 17 wherein the antiviralagent chosen from interferon α and ribavirin.
 21. A pharmaceuticalcomposition for treating or preventing a hepatitis viral C infectioncomprising administering at least one compound according to formula I asdefined in claim 1 together with at least one pharmaceuticallyacceptable carrier or excipient.
 22. A method for inhibiting or reducingthe activity of viral polymerase in a host comprising administering atherapeutically effective amount of at least one compound having theformula I:

or a pharmaceutically acceptable salts thereof, wherein: R is H, —NR₂R₃or OR₄ wherein R₂ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl,or C₂₋₆ alkynyl; R₃ isH or a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl; R₄ is H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl; and Ra is chosen from H, monophosphate,diphosphate, triphosphate, carbonyl substituted with a C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl and

wherein each Rc is independently chosen from H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and an hydroxy protecting group; wherein saidnucleoside is present in the form of the (−) enantiomer,(+) enantiomerand mixtures thereof, including racemic mixtures.
 23. The method ofclaim 22 wherein said viral polymerase is a HCV polymerase.
 24. A methodfor treating or preventing a Flaviviridea viral infection in a hostcomprising administering a therapeutically effective amount of at leastone compound having the formula II or a pharmaceutically acceptable saltthereof:

wherein Z is H, —NR′₂R_(3′) or OR_(4′) wherein R_(2′) is H, C₁₋₆ alkyl,C₂₋₆ alkenyl,or C₂₋₆ alkynyl; R_(3′) is H or a C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl; R_(4′)is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl; and Yis N or C—X; X is chosen from of H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, CN, CF₃, N₃, NO₂, C₆₋₁₀ aryl, C₆₋₁₀ heteroaryl and CORbwherein Rb is chosen from of H, OH, SH, C₁₋₆ alkyl, C₁₋₆ aminoalkyl,C₁₋₆ alkoxy and C₁₋₆ thioalkyl; and Ra is chosen from of H,monophosphate, diphosphate, triphosphate, carbonyl substituted with aC₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl, and

wherein each Rc are independently chosen from H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and an hydroxy protecting group, wherein saidcompound is in the form of a single enantiomer or a mixture thereofincluding racemic mixtures.
 25. The method of claim 24 wherein saidcompound of formula II of the present invention is substantially in theform of the (+) enantiomer.
 26. The method of claim 24 whereinsais-compound of formula II of the present invention is substantially inthe form of the (−) enatiomer.
 27. The method of claim 24 wherein Ra ischosen from H, monophosphate, diphosphate, and triphosphate.
 28. Themethod of claim 24 wherein Ra is chosen from monophosphate, diphosphate,and triphosphate.
 29. The method of claim 24 wherein Ra is triphosphate.30. The method of claim 24 wherein Ra is H.
 31. The method of claim 24wherein Y is C—X.
 32. The method of claim 24 wherein Y is C—X and X isH, methyl, or Halogen.
 33. The method of claim 24 wherein Y is C—X and Xis H, methyl or halogen.
 34. The method of claim 24 wherein Y is C—X andX is H, methyl or F.
 35. The method of claim 24 wherein Y is C—X and Xis H or F.
 36. The method of claim 24 wherein Y is C—X and X is H. 37.The method of claim 24 wherein Y is C—X and X is F.
 38. The method ofclaim 24 wherein Z is OH.
 39. The method of claim 24 wherein Z is NH₂.40. The method of claim 25 wherein Ra is chosen from H, monophosphate,diphosphate, and triphosphate.
 41. The method of claim 25 wherein Ra ischosen from monophosphate, diphosphate, and triphosphate.
 42. The methodof claim 25 wherein Ra is triphosphate.
 43. The method of claim 25wherein Ra is H.
 44. The method of claim 25 wherein Y is C—X.
 45. Themethod of claim 25 wherein Y is C—X and X is H, methyl, or Halogen. 46.The method of claim 25 wherein Y is C—X and X is H, methyl or halogen.47. The method of claim 25 wherein Y is C—X and X is H, methyl or F. 48.The method of claim 25 wherein Y is C—X and X is H or F.
 49. The methodof claim 25 wherein Y is C—X and X is H.
 50. The method of claim 25wherein Y is C—X and X is F.
 51. The method of claim 25 wherein Z is OH.52. The method of claim 25 wherein Z is NH₂.
 53. The method of claim 26wherein Ra is chosen from H, monophosphate, diphosphate, andtriphosphate.
 54. The method of claim 26 wherein Ra is chosen frommonophosphate, diphosphate, and triphosphate.
 55. The method of claim 26wherein Ra is triphosphate.
 56. The method of claim 26 wherein Ra is H.57. The method of claim 26 wherein Y is C—X.
 58. The method of claim 26wherein Y is C—X and X is H, methyl, or Halogen.
 59. The method of claim26 wherein Y is C—X and X is H, methyl or halogen.
 60. The method ofclaim 26 wherein Y is C—X and X is H, methyl or F.
 61. The method ofclaim 26 wherein Y is C—X and X is H or F.
 62. The method of claim 26wherein Y is C—X and X is H.
 63. The method of claim 26 wherein Y is C—Xand X is F.
 64. The-method of claim 26 wherein Z is OH.
 65. The methodof claim 26 wherein Z is NH₂.
 66. The method of claim 24 wherein thecompound of formula II is(+)Cis-2-hydroxymethyl-4-(cytosin-1′-yl)-1,3-Dioxolane (Compound #17(+)).
 67. The method of claim 24 wherein the compound of formula II is(+)Cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-Dioxolane(Compound #18 (+)).
 68. The method of claim 24 wherein the compound offormula II is(−)Cis-2-hydroxymethyl-4-(5′-fluorocytosin-1′-yl)-1,3-Dioxolane(Compound #18 (−)).
 69. The method of claim 24 wherein the compound offormula II is(+)Cis-2-hydroxymethyl-4-(5′-azacytosin-1′-yl)-1,3-Dioxolane (Compound#19 (+)).
 70. The method of claim 24 wherein the compound of formula IIis(+)-cis-2-hydroxymethyl-4-(5′-methylcytosin-1′-yl)-1,3-dioxolane(β-D-)(compound#20) (+)
 71. The method of claim 24 wherein the compound offormula II is (+)-cis-2-hydroxymethyl-4-(N-1′-thiminyl)-1,3-dioxolane(-D-) (compound#21) (+)
 72. The method of claim 24 wherein saidFlaviviridea viral infection is hepatitis C.
 73. A method for treatingor preventing a hepatitis C viral infection in a host comprisingadministering to the host a therapeutically effective amount of at leastone compound according to formula II as defined in claim 24 and at leastone further antiviral agent.
 74. The method according to claim 73wherein the further antiviral agent is chosen from of viral serineprotease inhibitor, viral polymerase inhibitor and viral helicaseinhibitor.
 75. The method according to claim 73 wherein the antiviralagent is chosen from interferon (IFN), interferon αx-2a, interferonα-2b, consensus interferon (CIFN), ribavirin, amantadine, rimantadine,interleukine-12, ursodeoxycholic acid (UDCA), glycyrrhizin and silybummarianum.
 76. The method according to claim 73 wherein the antiviralagent chosen from interferon α and ribavirin.
 77. A pharmaceuticalcomposition for treating or preventing a hepatitis viral C infectioncomprising administering at least one compound according to formula IIas defined in claim 73 together with at least one pharmaceuticallyacceptable carrier or excipient.
 78. A method for inhibiting or reducingthe activity of viral polymerase in a host comprising administering atherapeutically effective amount of at least one compound having theformula II or a pharmaceutically acceptable salt thereof:

wherein Z is H, —NR_(2′)R_(3′) or OR_(4′) wherein R_(2′) is H, C₁₋₆alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl; R_(3′) is H or a C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl; R_(4′)is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl; and Y is N or C—X; X is chosen from of H, halogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, CN, CF₃, N₃, NO₂, C₆₋₁₀ aryl, C₆₋₁₀heteroaryl and CORb wherein Rb is chosen from of H, OH, SH, C₁₋₆ alkyl,C₁₋₆ aminoalkyl, C₁₋₆ alkoxy and C₁₋₆ thioalkyl; and Ra is chosen fromof H, monophosphate, diphosphate, triphosphate, carbonyl substitutedwith a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl, and

wherein each Rc are independently chosen from H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and an hydroxy protecting group, wherein saidcompound is in the form of a single enantiomer or a mixture thereofincluding racemic mixtures.
 79. The method of claim 78 wherein saidviral polymerase is a HCV polymerase.